I. Gene Therapy
Gene therapy is an approach to treating disease, generally human disease, that is based on the modification of gene expression in cells of the patient. It has become apparent over the last decade that the single most outstanding barrier to the success of gene therapy as a strategy for treating inherited diseases, cancer, and other genetic dysfunctions is the development of useful gene transfer vehicles.
Eukaryotic viruses have been employed as vehicles for somatic gene therapy. Among the viral vectors that have been cited frequently in gene therapy research are adenoviruses. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. Human adenoviruses are composed of a linear, approximately 36 kb double-stranded DNA genome, which is divided into 100 map units (m.u.), each of which is 360 bp in length. The DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication. The gene products are organized into early (E1 through E4) and late (L1 through L5) regions, based on expression before or after the initiation of viral DNA synthesis [see, e.g., Horwitz, Virology, 2d edit., ed. B. N. Fields, Raven Press, Ltd., New York (1990)].
Recombinant adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are currently being developed for gene therapy. Both Ad2 and Ad5 belong to a subclass of adenovirus and are not associated with human malignancies.
Recombinant adenoviruses are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. High titers (10.sup.13 plaque forming units/ml) of recombinant virus can be easily generated in an adenovirus-transformed, human embryonic kidney cell line 293 [ATCC CRL1573]. The 293 cell line contains a functional adenovirus E1a gene which provides a transacting E1a protein. It can be cryo-stored for extended periods without appreciable losses.
The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders [K. F. Kozarsky et al, Somatic Cell Mol. Genet., 19:449-458 (1993) ("Kozarsky et al I"); K. F. Kozarsky et al, J. Biol. Chem., 269:13695-13702 (1994) ("Kozarsky et al II); Y. Watanabe, Atherosclerosis, 36:261-268 (1986); K. Tanzawa et al, FEBS Letters, 118(1):81-84 (1980); J. L. Golasten et al, New Engl. J. Med., 309:288-296 (1983); S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993); and S. Ishibashi et al, J. Clin. Invest., 93:1885-1893 (1994)]. Indeed, a recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved for use in at least two human CF clinical trials [see, e.g., J. Wilson, Nature, 365:691-692 (Oct. 21, 1993)]. The use of adenovirus vectors in the transduction of genes into hepatocytes in vivo has previously been demonstrated in rodents and rabbits [see, e.g., Kozarsky II, cited above, and S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993)]. Further support of the safety of recombinant adenoviruses for gene therapy is the extensive experience of live adenovirus vaccines in human populations.
However, many humans have pre-existing immunity to human adenoviruses as a result of previous natural exposure, and this immunity is a major obstacle to the use of recombinant human adenoviruses for gene therapy protocols.
II. Vaccines
Replication competent, recombinant adenovirus (Ad) containing a variety of inserted genes have been used as vaccine compositions with some success [see, e.g. Davis, U.S. Pat. No. 4,920,309]. Others have described the insertion of a foreign gene into a live [L. Prevac, J. Infect. Dis., 161:27-30 (1990)] and a replication-defective adenovirus for putative use as a vaccine [See, e.g. T. Ragot et al, J. Gen. Virol., 74:501-507 (1993); M. Eliot et al, J. Gen. Virol., 71:2425-2431 (1990); and S. C. Jacobs et al, J. Virol., 66:2086-2095 (1992)]. Jacobs et al, cited above, describes a recombinant E1-deleted, E3 intact, Ad containing encephalitis virus protein NS1 under the control of a heterologous cytomegalovirus (CMV) promoter. When mice were immunized with the recombinant Ad vaccines and challenged with virus, Jacobs et al obtained partial protection (at most a 75% protection) for an average survival of 15 days. Eliot et al, cited above, describe a recombinant E1-deleted, partially E3-deleted Ad with pseudorabies glycoprotein 50 inserted into the E1 deletion site under the control of a homologous Ad promoter. In rabbits and mice, after immunization and challenge, only partial protection was obtained (i.e., about one-third). Ragot et al, cited above, describe a recombinant E1-deleted, partially E3-deleted Ad with Epstein Barr virus glycoprotein gp340/220 inserted into the E1 deletion site under the control of a homologous Ad promoter. In marmosets (tamarins) after three high dose (5.times.10.sup.9 pfu, 1.times.10.sup.10 pfu and 2.times.10.sup.10 pfu), intramuscular immunizations and viral challenge, full protection was obtained.
For certain highly infectious diseases, there is a demand for an effective vaccine. Desirably, a vaccine should be effective at a low dosage to control the occurrence of side effects or to enable sufficient amounts of vaccine to be introduced into the animal or human.
There exists a need in the gene therapy art for the development of additional adenovirus vector constructs that do not stimulate immediate immune responses which quickly eliminate the recombinant virus and the therapeutic transgene from the patient. There also exists a need in the vaccine art for new vaccine carriers, which are safe and effective in humans and other mammals.